1. Field of the Invention
The present invention relates to an immunoassay for determining the relative concentrations of two different immunogenic analytes. In particular, the invention relates to an assay for determining relative concentrations, in a single sample, of related hormonal metabolites such as pregnanediol-3-glucuronide (P-3-G) and estrone-3-glucuronide (E.sub.1 -3-G). Even more particularly, the invention relates to an immunoassay suitable for testing for constituents in human urine to determine the human fertile period, that is, the period in which viable sperm and a viable ovum may be present simultaneously in the female reproductive tract.
2. The Prior Art Background
For a number of reasons it may be clinically and/or diagnostically desirable to determine the relative concentrations of two separate immunogenically reactive analytes in a single sample. In some instances, mammalian hormonal activity and/or metabolism creates situations where the relationships between concentrations of hormones or metabolites in body fluids are chronologically related to other events. In particular, the relative concentrations of P-3-G and E.sub.1 -3-G in human urine may be used to ascertain the fertile period of the menstrual cycle. And since, for a variety of reasons, contraceptive devices and materials may not be available for use, techniques such as immunoassays for determining the fertile period of the menstrual cycle have become desirable.
The human menstrual cycle is governed by the cyclical release of hormones from the female glands and organs. Such release is predictable and specifically related to ovulation by which ova are released from the ovaries and the lining of the uterus is made ready for pregnancy. Eventually, the released hormones and/or metabolites thereof find their way into the urine. The specific biological phenomena are described in detail and with clarity in European Patent Publication No. 0086095, published Aug. 17, 1983 in European Patent Office Bulletin 83/33. And suffice it to say, that during a normal menstrual cycle, the level of E.sub.1 -3-G in female urine begins to rise about 6 days prior to ovulation, reaches its peak about 1 day before ovulation and falls rapidly during and after ovulation. The level of P-3-G in female urine begins to rise on the day of ovulation, reaches its peak 2 to 3 days after ovulation and remains elevated for the duration of the luteal phase. The relationships between P-3-G and E.sub.1 -3-G levels are known, and from the '095 European patent publication identified above, the ratio of estrogen metabolites to progestin metabolites in the urine has been found to be useful in monitoring the progress of the menstrual cycle.
Of particular importance in following the menstrual cycle by determining hormonal activity is the fact that during the most fertile period, the level of E.sub.1 -3-G in the urine is approximately 20 times the level of P-3-G or greater. Thus, a simple and reliable assay capable of determining and/or detecting that period of time when the E.sub.1 -3-G/P-3-G ratio is 20 or greater would be extremely valuable in determining whether the female is fertile.
In the '095 patent publication identified above, an immunoassay procedure is disclosed for determining the relative concentrations of antigens in a single sample. The immunoassay employs a dual ligand molecule comprising two different antigens irreversibly bound together through a bridge support molecule to present an elongated ligand molecule having an antigenic moiety at one end and a different antigenic moiety at the other end. Bridge length is said to be crucial to the avoidance of steric hinderance and it has been found that the assay itself is sensitive to the dual ligand concentration.
The assay described in the '095 patent publication was said to be an improvement over the assay described in British Patent Specification No. GB 2029011B which includes a synthetic bifunctional ligand prepared by coupling two different antigenic moieties through a protein linkage such as bovine serum albumin (BSA). However, the number of molecules of each steroid per molecule of BSA may be independently varied by adjusting the stoichiometry of the reagents and calibration remains difficult. And as outlined in the '095 disclosure, the procedure of the '011B patent specification does not always provide for sensitive assay results because of the tendency of the bifunctional ligand to form multivalent immunocomplexes and to react nonspecifically.